Conjugate complexes for analyte detection

Abstract

The present invention relates to novel conjugate complexes for immunoassays as well as kits comprising these conjugate complexes, methods of producing these complexes, and methods of detecting an analyte by use of these complexes. The conjugate complexes of the invention comprise one or more non-nucleic acid receptors capable of specifically binding an analyte, one or more nucleic acid markers comprising a predetermined nucleotide sequence, one or more first linker molecules capable of specifically binding the non-nucleic acid receptor and the nucleic acid marker, and one or more second linker molecules capable of specifically binding the first linker molecules.

We claim: 
     
       1. A conjugate complex for binding to an analyte of interest, comprising:
 (a) at least two non-nucleic acid receptors that specifically bind the analyte; 
 (b) at least two nucleic acid markers comprising a predetermined nucleotide sequence; 
 (c) at least two first linker molecules, wherein each linker molecule specifically binds the non-nucleic acid receptor and the nucleic acid marker; and 
 (d) at least two second linker molecules that specifically binds the first linker molecules, 
 wherein the at least two non-nucleic receptors and the at least two nucleic acid markers are each coupled to one or more binding partners of the first linker molecule to facilitate binding of the at least two non-nucleic receptors and the at least two nucleic acid markers to the at least two first linker molecules, 
 wherein the at least two second linker molecules are each coupled to one or more binding partners of the first linker molecule to facilitate binding of the at least two second linker molecules to the at least two first linker molecules, 
 wherein the at least two first linker molecules and the at least two second linker molecules are at least bivalent. 
 
     
     
       2. A conjugate complex according to  claim 1 , wherein the binding of the at least two first linker molecules to the non-nucleic acid receptor, the nucleic acid marker and/or the second linker molecule is non-covalent. 
     
     
       3. A conjugate complex according to  claim 1 , wherein the at least two second linker molecules are organic polymers, proteins, polysaccharides, or mixtures thereof. 
     
     
       4. A conjugate complex according to  claim 1 , wherein the at least two second linker molecules are organic polymer molecules selected from the group of linear or branched polyethyleneimines, polyacrylamides, polyamines, and polyamidoamines. 
     
     
       5. A conjugate complex according to  claim 1 , wherein the at least two second linker molecules are proteins selected from the group consisting of albumines, immunoglobulins and poly-amino acids. 
     
     
       6. A conjugate complex according to any one of  claim 1 , wherein the at least two second linker molecules are polysaccharides selected from the group of linear dextrans, cyclic dextrans and branched dextrans. 
     
     
       7. A conjugate complex according to  claim 1 , wherein the at least two second linker molecules are nucleic acids selected from the group consisting of nucleic acid oligomers consisting of two complementary nucleic acid strands, wherein each of the complementary nucleic acid strands binds a first linker molecule, and polynucleotides. 
     
     
       8. A conjugate complex according to  claim 1 , wherein at least two second linker molecules are nucleic acid oligomers consisting of two complementary nucleic acid strands, wherein the complementary nucleic acid strands binds the first linker molecule. 
     
     
       9. A conjugate complex according to  claim 1 , wherein the at least two second linker molecules are a combination of two or more different molecules selected from the group consisting of linear, branched or dendritic polyethyleneimines, polyacrylamides, polyamines, polyamidoamines, albumines, immunoglobulins, poly-amino acids, linear dextrans, cyclic dextrans, branched dextrans, polynucleotides, and nucleic acid oligomers consisting of two complementary nucleic acid strands. 
     
     
       10. A conjugate complex according to  claim 1 , wherein the at least two non-nucleic receptors and the at least two nucleic acid markers are each coupled to at least two binding partners of the first linker molecule that facilitate binding of the at least two non-nucleic receptors and the at least two nucleic acid markers to the at least two first linker molecules. 
     
     
       11. A conjugate complex according to  claim 10 , wherein the at least two binding partners of the first linker molecule coupled to the at least two non-nucleic acid receptors and the one or more nucleic acid markers are the same or different. 
     
     
       12. A conjugate complex according to  claim 10 , wherein the at least two binding partners of the first linker molecule are covalently coupled to the at least two non-nucleic acid receptors and the nucleic acid markers. 
     
     
       13. A conjugate complex according to  claim 1 , wherein the at least two second linker molecules are each coupled to at least two binding partners of the first linker molecule. 
     
     
       14. A conjugate complex according to  claim 13 , wherein the at least two binding partners of the first linker molecule are covalently coupled to the at least two second linker molecules. 
     
     
       15. A conjugate complex according to  claim 10 , wherein the binding of the at least two binding partners of the first linker molecule to the first linker molecule is non-covalent. 
     
     
       16. A conjugate complex according to  claim 10 , wherein the first linker molecule is avidin or streptavidin or a biotin-binding fragment or mutant thereof. 
     
     
       17. A conjugate complex according to  claim 10 , wherein the binding partner of the first linker molecule is biotin or a biotin analog. 
     
     
       18. A conjugate complex according to  claim 1 , wherein the non-nucleic receptor is an antibody or antibody fragment. 
     
     
       19. A conjugate complex according to  claim 1 , wherein the analyte is an antigen or hapten. 
     
     
       20. A conjugate complex according to  claim 1  further comprising at least two modulators that specifically bind to the at least two first linker molecules to saturate non-occupied binding sites of the first linker molecule for the non-nucleic acid receptor, the nucleic acid marker, the second linker molecule, or a binding partner of the first linker molecule. 
     
     
       21. A conjugate complex according to  claim 20 , wherein the at least two modulators are positively charged. 
     
     
       22. A conjugate complex according to  claim 20 , wherein the at least two modulators are amino-biotin, diamino-biotin or amino-substituted biotin analogs. 
     
     
       23. A conjugate complex according to  claim 1 , wherein the nucleic acid marker is dsDNA, ssDNA, dsRNA, ssRNA or a DNA:RNA hybrid. 
     
     
       24. A method for the preparation of a conjugate complex according to  claim 1 , comprising the steps of:
 (a) contacting at least two nucleic acid markers with at least two first linker molecules, wherein each first linker molecule specifically binds the at least two nucleic acid markers to form a complex of at least two nucleic acid markers with at least two first linker molecules; 
 (b) contacting the complex of step (a) with at least two non-nucleic acid receptors, wherein each non-nucleic acid receptor specifically binds the at least two first linker molecules to form a complex of at least two non-nucleic acid receptors, at least two nucleic acid markers and at least two first linker molecules; and 
 (c) contacting the complex of step (b) with at least two second linker molecules that specifically bind the at least two first linker molecules to form a complex of at least two non-nucleic acid receptors, at least two nucleic acid markers, at least two first linker molecules and at least two second linker molecules. 
 
     
     
       25. A method according to  claim 24 , further comprising the step of:
 (d) contacting the complex of step (c) with at least two modulators that specifically bind the at least two first linker molecules to saturate non-occupied binding sites of the first linker molecule for the non-nucleic acid receptor, the nucleic acid marker and the second linker molecule to form a complex of at least two non-nucleic acid receptors, at least two nucleic acid markers, at least two first linker molecules, at least two second linker molecules and at least two modulators. 
 
     
     
       26. A method for the preparation of a conjugate complex that binds an analyte of interest, comprising
 (i) at least two non-nucleic acid receptors that specifically binds the analyte; 
 (ii) at least two nucleic acid markers comprising a predetermined nucleotide sequence; 
 (iii) at least two first linker molecules, wherein each linker molecule specifically binds the non-nucleic acid receptor and the nucleic acid marker; 
 (iv) at least two nucleic acid oligomers that specifically bind at least two first linker molecules, wherein the at least two nucleic acid oligomers comprise two complementary nucleic acid strands distinct from the nucleic acid marker; and 
 (v) at least two organic polymers, polynucleotides, proteins or polysaccharides that specifically bind at least two first linker molecules, wherein the polynucleotides of (v) are distinct from the nucleic acid marker and the at least two nucleic acid oligomers; 
 wherein the method comprises the steps of: 
 (a) contacting one nucleic acid strand of the at least two nucleic acid oligomers with at least two first linker molecules to form a first conjugate of at least two first linker molecules and one nucleic acid strand of the at least two nucleic acid oligomers; 
 (b) contacting the nucleic acid strand of the at least two nucleic acid oligomers complementary to that used in step (a) with at least two first linker molecules to form a second conjugate of at least two first linker molecules and one nucleic acid strand of the at least two nucleic acid oligomers complementary to that used in step (a); 
 (c) contacting the conjugate of step (a) with at least two nucleic acid markers to form a first complex of at least two first linker molecules conjugated to one nucleic acid strand of the at least two nucleic acid oligomers and at least two nucleic acid markers; 
 (d) contacting the conjugate of step (b) with at least two non-nucleic receptors to form a second complex of at least two first linker molecules conjugated to one nucleic acid strand of the at least two nucleic acid oligomers complementary to that used in step (a) and (c) and at least two non-nucleic acid receptors; 
 (e) contacting the first complex of step (c) with at least two organic polymers, polynucleotides, proteins or polysaccharides, to form a third complex of at least two first linker molecules conjugated to one nucleic acid strand of the at least two nucleic acid oligomers, at least two nucleic acid markers and at least two organic polymers, polynucleotides, proteins or polysaccharides; and 
 (f) contacting the second complex of step (d) with the third complex of step (e) to form the complex conjugate of at least two first linker molecules conjugated to one nucleic acid strand of the at least two nucleic acid oligomers, at least two nucleic acid markers, at least two organic polymers, polynucleotides, proteins or polysaccharides, at least two first linker molecules conjugated to one nucleic acid strand of the at least two nucleic acid oligomers complementary to that used in step (a) and (c) and at least two non-nucleic acid receptors. 
 
     
     
       27. A method according to  claim 26 , wherein the method further comprises the steps of contacting the complexes of steps (d) and (e) with at least two modulators that specifically bind at least two first linker molecules before step (f). 
     
     
       28. A method for detecting an analyte in a sample, the method comprising the steps of:
 (a) contacting the conjugate complex of  claim 1  comprising at least two non-nucleic acid receptors that specifically bind said analyte with said sample to form a complex of said analyte and said conjugate complex; 
 (b) specifically detecting the presence of the at least two nucleic acid markers in said complex; 
 wherein the presence of the at least two nucleic acid markers indicates the present of the analyte in said sample. 
 
     
     
       29. A method according to  claim 28 , wherein the detecting step (b) comprises amplifying the at least two nucleic acid markers in a PCR reaction. 
     
     
       30. A kit comprising at least two conjugate complexes according to  claim 1 .